Phospholink nucleotides for sequencing applications

ABSTRACT

The present invention provides labeled phospholink nucleotides that can be used in place of naturally occurring nucleotide triphosphates or other analogs in template directed nucleic acid synthesis reactions and other nucleic acid reactions and various analyses based thereon, including DNA sequencing, single base identification, hybridization assays, and others.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional U.S. Patent ApplicationNo. 61/115,381, filed Nov. 17, 2008, the full disclosure of which isincorporated herein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

For nucleic acid analyses based upon detection of polymerase-mediatedincorporation of nucleotides, the label on the detectable nucleotidescan have a significant impact on the efficiency and accuracy of suchanalyses. Fluorophore-labeled nucleotides are generally used in suchnucleic acid analyses. However, traditional methods of labelingnucleotides with fluorophores can pose problems with respect to lack ofbrightness of the label, photodamage to the polymerase, and instabilityof the label due to photobleaching. In addition, suchfluorophore-labeled nucleotides can require the use of expensiveequipment, such as high power lasers, electron multiplying CCD cameras,and the like. As such, brighter, more robust labels for sequencingapplications are desirable.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides nucleotides labeled withbeads that produce a brighter signal than is generally achievable withtraditional methods of labeling nucleotides.

In one aspect, the invention provides a composition that includes alabeled phospholink nucleotide. In this aspect, the labeled phospholinknucleotide includes a structure that has formula:

wherein B is a nucleobase; S is a sugar moiety; L is a bead comprising afluorophore; R₁ is selected from oxygen and sulfur; R₂ is a linkermoiety; and n is an integer selected from 0 to 9.

In one aspect, the invention provides a composition comprising amicrofluidic flow cell. In a further aspect, the microfluidic flow cellincludes a substrate with a surface, and a nucleic acid polymerase isimmobilized on the surface of the substrate. In a still further aspect,the microfluidic flow cell further includes at least four differentiallylabeled nucleotides, wherein the labeled nucleotides include beadscomprising fluorophores.

In one aspect, the invention provides a method of determining anidentity of a nucleotide in a template nucleic acid sequence. Such amethod includes the steps of (i) providing the template nucleic acidsequence complexed with a polymerase enzyme capable of templatedependent synthesis of a complementary nascent sequence as a firstcomplex; (ii) contacting the first complex with a labeled phospholinknucleotide, wherein the labeled phospholink nucleotide includes a beadwith at least one fluorophore, and wherein the labeled phospholinknucleotide is complementary to a known nucleotide; and (iii) detectingwhether the labeled phospholink nucleotide is incorporated into thenascent sequence. In this aspect, incorporation of the labeledphospholink nucleotide is indicative that the complementary nucleotideis in a position in the template nucleic acid that is being processed bythe polymerase enzyme.

In one aspect, the invention provides a nucleic acid sequencing system.Such a system includes: (i) a body structure with a plurality ofmicrofluidic channels; (ii) a source of one or more template nucleicacids; (iii) a source of one or more sequencing reagents; and (iv) afluid flow controller that flows the template nucleic acid and the oneor more sequencing reagents into contact in the at least firstmicrofluidic channel. In a further aspect, the template nucleic acidsource is capable of being fluidly coupled to at least a first one ofthe microfluidic channels. In a still further aspect, the sequencingreagent source is capable of being fluidly coupled to the at least firstmicrofluidic channel. In a still further aspect, the source of the oneor more sequencing reagents includes a set of at least four differentlylabeled nucleotide analogs, and the labeled nucleotide analogs include afluorescent bead. In a further aspect, the nucleic acid sequencingsystem includes a detector. In a still further aspect, the nucleic acidsequencing system forms one or more sequencing products and the detectordetects the one or more sequencing products, thereby determining atleast a subsequence of the template nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary method for coupling a phospholinknucleotide to a bead.

FIG. 2 illustrates different bead-labeled phospholink nucleotides withlinkers of different length and structure. FIG. 2A illustrates abead-labeled phospholink nucleotide with an aminohexyl linker. FIG. 2Billustrates a bead-labeled phospholink nucleotide with a linkercomprising two aminohexyl groups. FIG. 2C illustrates a bead-labeledphospholink nucleotide with a linker comprising an aminohexyl linker anda chain of poly(ethylene glycol) groups.

FIG. 3 shows the results of a DNA incorporation experiment withbead-labeled phospholink nucleotides. The lanes marked A, B and C showthe results from DNA incorporation experiments using bead-labeledphospholink nucleotides with the structures in FIGS. 2A, 2B and 2Crespectively. The lanes marked AF, BF and CF are control lanes.

FIG. 4 shows the diffusion times of bead-labeled phospholink nucleotidesand unconjugated beads in zero-mode waveguides with diameters of (A) 80nm and (B) 120 nm.

FIG. 5 shows results from measurements of flowthrough absorbance. FIG.5A shows the absorbance at 265 nm of two different types of phospholinknucleotides as a function of number of washes, and FIG. 5B shows theabsorbance spectra of different bead-labeled phospholink nucleotidesafter 7 washes.

DETAILED DESCRIPTION OF THE INVENTION Overview

The present invention provides methods and compositions for sequencingapplications utilizing nucleoside polyphosphates labeled with detectablebeads. In one embodiment, the nucleotides of the invention are labeledwith fluorescent beads. Such beads provide an advantage over labelsgenerally used in the art, because instead of the normal 1:1 ratiobetween a fluorescent label and a nucleotide, fluorescent beads cancomprise multiple fluorophores, resulting in a much brighter signal forevery labeled nucleotide. A further advantage to such fluorescent beadscomprising multiple fluorophores is that the signal from such beads isunlikely to be reduced to background levels. Conventional organicfluorophores blink, which can introduce apparent insertion errors (falsepositives) in sequencing applications. Since beads comprising multiplefluorophores are highly likely to have at least one fluorophore alwaysemitting fluorescence, any reduction in signal that may occur due to onefluorophore blinking is averaged out over the multiple fluorophorescarried by a particular bead. The multiple fluorophores also reduce theeffects of photobleaching, because even if one fluorophorephotobleaches, the other fluorophores are likely to continue emitting asignal. Fluorescent beads comprising multiple fluorophores thus providea significantly improved signal relative to labels comprising only asingle fluorophore.

A further advantage of the fluorescent beads used in accordance with thepresent invention is that multiple nucleoside polyphosphates can beattached to these beads, which allows for “recycling” of these beadsthrough multiple incorporation events, because when one nucleosidepolyphosphate is incorporated into the nascent chain and thensubsequently cleaved from the bead, the remaining nucleosidepolyphosphates joined to the bead are still available for subsequentincorporation events. This also results in a reduction of the apparentbinding affinity between the nucleotide-bead conjugates to the enzyme,allowing lower background signal, and thus higher signal-to-noiseratios. In a further embodiment, the detectable beads are joined to eachnucleoside polyphosphate through a terminal phosphate of the nucleosidepolyphosphate.

Labeled Phospholink Nucleotides

The term “phospholink nucleotides” is generally used herein to refer tonucleoside polyphosphates that can be linked to a label through aterminal phosphate group. Unless otherwise noted, the term “phospholinknucleotide” and “nucleotide” are used interchangeably herein.

In one aspect, the invention provides a labeled phospholink nucleotidethat includes a structure according to the general formula:

in which B represents a natural or non-natural nucleobase or nucleobaseanalog; S is a sugar moiety, an acyclic moiety or a carbocyclic moiety;L is a detectable label, R₁ is selected from oxygen and sulfur, R₂ is alinker moiety, and n is an integer selected from 0 to 9. It should benoted that R₂ is not a required element of labeled phospholinknucleotide of the invention, and that the label “L” may also optionallyinclude a linker moiety.

In one embodiment, the linker group R₂ comprises one or more groups suchthat the compound represented by formula (I) is a nucleosidepolyphosphate analog. For example, R₂ may comprise one or morephosphonate groups, brominated phosphate groups, and the like.

The nucleobase represented by “B” in Formula (I) can be selected fromany of the natural or non-natural nucleobases or nucleobase analogs,including e.g., purine or pyrimidine bases that are routinely found innucleic acids and nucleic acid analogs, including adenine, thymine,guanine, cytidine, uracil, and in some cases, inosine. The nucleobasesof the present invention may include the conventional bases describedherein or they may include such bases substituted at one or more sidegroups, or other analogs, such as 1, N-6-ethenoadenosine or pyrrolo C,in which an additional ring structure renders the nucleobase neither apurine nor a pyrimidine.

The S group of Formula (I) can be a sugar moiety that provides asuitable backbone for synthesizing a nucleic acid strand. In one aspect,the sugar moiety is selected from D-ribosyl, 2′ or 3′ D-deoxyribosyl,2′3′-D-dideoxyribosyl, 2′,3′-D-didehydrodideoxyribosyl, 2′ or 3′aminoribosyl, 2′ or 3′ mercaptoribosyl, 2′ or 3′ alkothioribosyl,acylcic, carbocyclic, or other modified sugar moieties. In a furtheraspect, a variety of carbocyclic or acyclic moieties can be incorporatedinto the S group in place of a sugar moiety, including e.g., thosedescribed in published U.S. Pat. No. 7,041,812, which is incorporatedherein by reference in its entirety for all purposes, and in particularfor its teachings regarding alternative moieties that can be used inplace of a sugar moiety.

As discussed further herein, “L” in Formula (I) is a detectable label.In one aspect, L is a bead comprising one or more detectable moieties,including without limitation: luminescent, chemiluminescent,fluorescent, fluorogenic, chromophoric, magnetic, light scattering,and/or chromogenic moieties.

Further details regarding the detectable label “L” and the linker moiety“R” are provided herein.

Bead-Labeled Phospholink Nucleotides

In one aspect, the present invention provides phospholink nucleotideslabeled with beads. In one embodiment, such beads comprise fluorophores.In further embodiments, the fluorophores are embedded in a materialcontained in or associated with the beads. Although for clarity's sakethe following embodiments of the invention are described in terms ofnucleotides linked to fluorescent beads, it will be appreciated that thepresent invention also encompasses nucleotides linked with beadscomprising other kinds of detectable labels, including withoutlimitation dyes (including surface-bound dyes on metal nanoparticleswith or without fluorescent enhancements), chromophores, enzymes,antigens, heavy metals, magnetic probes, phosphorescent groups,radioactive materials, chemiluminescent moieties, scattering orfluorescent nanoparticles, fluorescein labels, rhodamine labels, cyaninelabels (i.e., Cy3, Cy5, and the like, generally available from theAmersham Biosciences division of GE Healthcare), the Alexa Fluor® familyof fluorescent dyes (available from Invitrogen, Inc.) and otherfluorescent and fluorogenic dyes. Such labels are known in the art andare disclosed for example in Prober, et. al., Science 238: 336-41(1997); Connell et. al., BioTechniques 5(4): 342-84 (1987); Ansorge, et.al., Nucleic Acids Res. 15(11): 4593-602 (1987); and Smith et. al.,Nature 321:674 (1986), which are hereby incorporated by reference intheir entirety for all purposes and in particular for their teachingsregarding such labels.

It will be appreciated that the term “bead” as used herein refers to anyparticle that can be joined to phospholink nucleotides according to thepresent invention. Such beads include without limitation latex beads,glass beads, polymeric beads, metal nanoparticles, magneticnanoparticles, and avidin particles. Beads of the invention may furtherinclude without limitation inorganic materials, such as semiconductornanoparticles, including e.g., II-V and II-VI core shell nanocrystalsand the like. As will be appreciated, although the term bead encompassesspherical objects, any shape and size of bead can be used in accordancewith the present invention.

In one embodiment, nucleotides are joined to beads of diameters rangingfrom about 5 nm to about 1 μm. In a further embodiment, beads of use inthe invention have diameters ranging from about 10 nm to about 500 nm,from about 15 nm to about 400 nm, from about 20 nm to about 300 nm, fromabout 30 nm to about 200 nm, from about 40 nm to about 100 nm, and fromabout 50 nm to about 75 nm. Beads of the invention will generally bespherical in shape, but beads of other shapes are also encompassed bythe present invention.

In a further aspect, bead-labeled phospholink nucleotides of theinvention are encapsulated in order to block damage to the fluorophoresin the beads from radicals that can be generated during illuminatedreactions. The beads may be encapsulated in organic or inorganicmaterials, including, e.g., latex, polymeric materials such aspolyethylene glycols, alginates, and the like, and other matrices. Suchencapsulation can increase the read-length of sequencing reactionsutilizing such bead-labeled phospholink nucleotides by alleviating andpreventing photodamage, particularly the detrimental effects from freeradicals that can be generated from illumination of fluorophores. Inparticular, encapsulation of fluorophores in beads and then optionallyfurther encapsulation of the beads in latex, polymers, and/or othermatrices, isolates those fluorophores from the environment and preventfree radicals, including free radical oxygen species, that can causedamage to reactants in a sequencing reaction, including polymerases usedin sequencing-by-synthesis reactions. By limiting damage to polymerases,the read length of sequencing reactions utilizing such polymerases canbe increased.

Although for clarity's sake much of the discussion herein will be withrespect to beads comprising fluorophores, it will be appreciated thatthe methods and compositions of the present invention are not limited tofluorescent beads and encompass any bead with any detectable label, asdescribed further above.

Fluorescent Beads Comprising Multiple Fluorophores

Fluorescent beads for use in the present invention generally comprisemultiple fluorophores. Such beads will generally provide a brighter andmore robust signal than is possible with conventional fluorescentlabels. Conventional organic fluorophores blink, which can decrease thesignal and introduce apparent insertion errors when used in sequencingapplications. In contrast, the signal from beads carrying multiplefluorophores will not show a decreased signal due to blinking, becausethere is an increased likelihood that at least one fluorophore isemitting fluorescence at all times.

Fluorescent beads comprising multiple fluorophores also provide a morerobust signal than is possible with conventional fluorophore labels,because the signal of such beads is resistant to the effects ofphotobleaching. Photobleaching is an exponential process with respect tothe time a fluorophore is emitting light, which means that somefluorophores photobleach before enough photons have been emitted toallow them to be detected reliably. Beads containing multiplefluorophores alleviate or eliminate this problem, because it is likelythat at least one fluorophore will not photobleach and will be able toemit a signal.

In one exemplary embodiment, all of the fluorophores contained in beadsof the invention have the same color.

In one exemplary embodiment, a bead of the invention will comprisedifferent types of fluorophores (i.e., different colors). Such beadsallow mixing of colors in sequencing applications, which has theadvantage of requiring fewer separation channels. For example, a set ofbead-labeled nucleotides may include beads with only green dyes, beadswith only red dyes, and beads with half green/half red (which wouldappear yellow). Such a set of beads would reduce the number of channelsneeded for detection to just 2, because the three different kinds ofbeads would give (0,1), (1,0) and (½,½) signal strength for the threebeads respectively. A separate detection channel would thus not beneeded for each fluorophore color.

Attachment of Phospholink Nucleotides to Beads

In one embodiment, beads of use in the invention comprise surfacesderivatized with functional groups. In a further embodiment, suchfunctional groups are activated and then subsequently coupled to anamino-terminated nucleotide or nucleotide analog. Non-limiting examplesof functional groups of use in beads of the invention include carboxyl,amine, sulfate, aldehyde and thiol groups.

An exemplary mechanism for coupling a bead (also referred to herein as a“nanosphere” or “particle”) to a phospholink nucleotide is illustratedin FIG. 1. As shown in FIG. 1, a bead with a carboxyl-terminated surfacecan be activated by 1-Ethyl-3-(3-dimethylaminopropyl) caroboiimide andthen subsequently coupled to an aminoterminated nucleotide analog, whichin this illustration is a nucleotide hexaphosphate. Although a singlephospholink nucleotide is shown coupled to the bead in FIG. 1, it willbe appreciated that numerous phospholink nucleotides can be similarlycoupled to the same bead through this and similar methods.

In another exemplary embodiment, nucleotides joined to biotin are joinedto avidin particles through an interaction between biotin and avidin,thus coupling the nucleotide to the particle. Multiplebiotin-phospholink nucleotides can be attached to avidin particles. Suchbiotin-avidin linkages are well known and characterized in the art.

Linkers

In a further aspect, the phospholinked nucleotides of the inventioncomprise a nucleotide joined to a bead through a linker (represented bythe group “R₂” in Formula (I) provided above). Those of skill in the artwill appreciate that a linker can be of any form that is suitable tobind to the bead and to the nucleoside polyphosphate, thereby “linking”the two molecules together. It will be appreciated that linkers of usein the invention are linkers that provide an adequate distance betweenthe nucleotide and the bead to avoid interactions between the bead and apolymerase. As the bead is generally of larger size than a polymerase,the bead could hinder incorporation of the labeled nucleotide during anucleic acid synthesis reaction if it were too close to the polymerase.

Generally, a linker will be formed from a molecule comprising reactivefunctional groups that are complementary to functional groups on thesurface of the bead and/or the nucleoside polyphosphate, thereby formingthe necessary bonds. As used herein, the term “linker” and “linkermoiety” are used interchangeably.

In an exemplary aspect, linkers of the invention can be selected fromsubstituted or unsubstituted alkyl (such as alkane or alkene linkers offrom about C20 to about C30), substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocycloalkyl. In a further aspect, linkers of theinvention may include poly(ethylene glycol) (PEG) groups, saturated orunsaturated aliphatic structures comprised of single or connected rings,amino acid linkers, peptide linkers, nucleic acid linkers, PNA, LNA, aswell as linkers containing phosphate or phosphonate groups. Examples ofsome of these linker types are described in e.g., Provisional U.S.Patent Application No. 61/069,247, filed Mar. 13, 2008, which isincorporated herein by reference in its entirety for all purposes and inparticular for all teachings related to linkers.

As illustrated in FIG. 2, a variety of linkers can be used in accordancewith the invention. The structures in FIG. 2 are exemplary and are notmeant to be limiting as to the linking moieties that can be used inaccordance with the invention. Such linkers may include aminohexylgroups (FIGS. 2A and B) and/or chains of poly(ethylene glycol) groups(FIG. 2C). In a further embodiment, a combination of structures such asthose illustrated in FIG. 2 are used as linkers in accordance with theinvention.

In one aspect of the invention, an appropriate linker for a sequencingapplication is identified through assays for phospholink nucleotideincorporation in bulk DNA synthesis assays. The results of one suchassay are shown in FIG. 3. Among those tested, the linker that allowedsuccessful incorporation of the phospholink nucleotide (as shown in lane“C”) was the linker that contained a chain comprising multiplepoly(ethylene glycol) moieties. The structure of this linker isillustrated in FIG. 2C. The other two linkers illustrated in FIGS. 2Aand B were not successful in allowing incorporation of the labelednucleotide (see lanes A and B of FIG. 3).

In one embodiment, the linker is a member selected from substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocycloalkyl. In one example, the linker is selectedfrom straight- and branched carbon-chains, optionally including at leastone heteroatom (e.g., at least one functional group, such as ether,thioether, amide, sulfonamide, carbonate, carbamate, urea and thiourea),and optionally including at least one aromatic, heteroaromatic ornon-aromatic ring structure (e.g., cycloalkyl, phenyl).

The linker as a whole may comprise a single covalent bond or a series ofstable bonds. Thus, a reporter molecule (such as a fluorescent bead) maybe directly attached to another reactant, such as a nucleosidepolyphosphate, or the reporter molecule may be attached to a nucleosidepolyphosphate through a series of stable bonds. A linker that is aseries of stable covalent bonds can incorporate non-carbon atoms, suchas nitrogen, oxygen, sulfur and phosphorous, as well as other atoms andcombinations of atoms, as is known in the art.

If the linker is not directly attached to a phospholink nucleotideand/or a bead by a single covalent bond, the attachment may comprise acombination of stable chemical bonds, including for example, single,double, triple or aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds,phosphorus-nitrogen bonds, and nitrogen-platinum bonds.

Removal of Non-Labeled Nucleotides

In a further aspect of the invention, nucleotides that are notsuccessfully joined to a bead are separated from the bead-labelednucleotides using methods known in the art, including dialysis, HPLC,membrane filtration and size exclusion chromatography. Removing thenon-labeled nucleotides avoids having such nucleotides incorporated intoa nascent strand during a polymerase-mediate nucleic acid synthesisreaction, thus avoiding a missed incorporation events in sequencingapplications based on detecting such incorporated nucleotides.

In one exemplary embodiment, nucleotides that are not joined to a beadare removed by centrifugation through a membrane which is sized topermit the non-labeled nucleotides to pass through, whereas thebead-labeled nucleotides are retained. In an exemplary embodiment, a 100kDa MW cutoff membrane is used to remove non-labeled nucleotides beforethe labeled phospholink nucleotides are used in sequencing applications.In a further exemplary embodiment, the purification of labeledphospholink nucleotides can be monitored by measuring the absorbance ofthe flowthrough after several cycles of washes and centrifugationthrough a 100 kDa MW cutoff membrane.

In another exemplary embodiment, a phosphodiesterase enzyme is used toremove nucleotides that are not joined to beads according to theinvention. The phosphodiesterase will break phosphodiester bonds.Nucleotides joined to beads can be protected from the action of thephosphodiesterase through encapsulation, or separation by a membranewith a molecular weight cutoff that only lets nucleotides, but notbeads, diffuse through. In particular, reaction systems may be equippedwith partitioned regions that contain in one region a nucleic acidsynthesis complex and bead bound nucleotides, while in the other regionis disposed a phophodiesterase enzyme. The two regions are separated bya semipermeable barrier, e.g., a membrane or the like, that allows forfree diffusion of free nucleotides into the region containing thephosphodiesterase, but does not allow those nucleotides that remainbound to the beads to enter this region, or allow the phosphodiesteraseto enter the region in which the synthesis complex and bead boundnucleotides are disposed. The result is that bead bound nucleotidesremain within the reaction region occupied by the synthesis complex,while unbound nucleotides freely diffuse into the phophodiesteraseregion and are consumed by the enzyme. The phosphodiesterase enzyme maybe used in combination with the washing/centrifugation methods describedabove for removing nucleotides not joined to beads.

In another exemplary embodiment, excess unbound nucleotides are removedby dialysis methods. Such methods are well known in the art.

Sequencing Applications Utilizing Phospholink Nucleotides

Phospholink nucleotides of the invention are particularly useful insequencing applications, particularly so-called “single molecule”sequencing applications. Bead-labeled phospholink nucleotides provideseveral advantages over traditional methods of labeling nucleotides,including elimination and/or alleviation of photodamage, and increasingaccuracy by increasing label brightness

Single molecule sequencing applications are well known and wellcharacterized in the art. See, e.g., Rigler, et al., DNA-Sequencing atthe Single Molecule Level, Journal of Biotechnology, 86(3): 161 (2001);Goodwin, P. M., et al., Application of Single Molecule Detection to DNASequencing. Nucleosides & Nucleotides, 16(5-6): 543-550 (1997); Howorka,S., et al., Sequence-Specific Detection of Individual DNA Strands usingEngineered Nanopores, Nature Biotechnology, 19(7): 636-639 (2001);Meller, A., et al., Rapid Nanopore Discrimination Between SinglePolynucleotide Molecules, Proceedings of the National Academy ofSciences of the United States of America, 97(3): 1079-1084 (2000);Driscoll, R. J., et al., Atomic-Scale Imaging of DNA Using ScanningTunneling Microscopy. Nature, 346(6281): 294-296 (1990).

Other methods of single molecule sequencing known in the art includedetecting individual nucleotides as they are incorporated into a primedtemplate, i.e., sequencing by synthesis. Such methods often utilizeexonucleases to sequentially release individual fluorescently labelledbases as a second step after DNA polymerase has formed a completecomplementary strand. See Goodwin et al., “Application of SingleMolecule Detection to DNA Sequencing,” Nucleos. Nucleot. 16: 543-550(1997).

The present invention applies equally to sequencing all types of nucleicacids (DNA, RNA, DNA/RNA hybrids etc.) using a number of polymerizingenzymes (DNA polymerases, RNA polymerases, reverse transcriptases,mixtures, etc.). Therefore, appropriate nucleotide analogs serving assubstrate molecules for the nucleic acid polymerizing enzyme can consistof members of the groups of dNTPs, NTPs, modified dNTPs or NTPs, peptidenucleotides, modified peptide nucleotides, or modified phosphate-sugarbackbone nucleotides.

As discussed herein, the brighter and more robust signals provided bythe bead-labeled phospholink nucleotides of the invention reduce thenumber of false negatives in a sequencing reaction. In addition, thebrighter signals can enable the use of a faster camera, which canincrease the accuracy of detection in such sequencing reactions. Theshorter residence time events can be more accurately detected withfaster cameras, particularly since the brighter signal allows theshorter residence time events to be detected over background.

Template-Dependent Nucleic Acid Synthesis

The present invention is of particular use in sequencing methods basedon template-dependent nucleic acid synthesis. In such methods, one ormore nucleotides are introduced to a template primer complex in thepresence of polymerase. Template-dependent nucleotide incorporationtakes place as the primer (also referred to herein as the “nascentstrand”) is elongated. Such nucleotides are generally labeled, and eachnucleotide incorporated into the nascent strand can be detected usingthat label.

During or after each labeled nucleotide is added to the sequencingprimer, the nucleotide added to the sequencing primer is identified.This is most generally achieved by giving each nucleotide a differentdistinguishable label. By detecting which of the different labels areadded to the sequencing primer, the corresponding nucleotide added tothe sequencing primer can be identified and, by virtue of itscomplementary nature, the base of the target nucleic acid which thenucleotide complements can be determined. Once this is achieved, it isno longer necessary for the nucleotide that was added to the sequencingprimer to retain its label. In fact, the continued presence of labels onnucleotide complementing bases in the target nucleic acid that havealready been sequenced would very likely interfere with the detection ofnucleotide analogs subsequently added to the primer. Accordingly, labelsadded to the sequencing primer are generally removed after they havebeen detected. This preferably takes place before additional nucleotidesare incorporated into the oligonucleotide primer.

The labeled phospholink nucleotides of the present invention are ofparticular use in such sequencing-by-synthesis applications, because, asdiscussed further herein, the fluorescent beads used in the inventionprovide a much brighter signal than is possible with traditional methodsof labeling nucleotides for such applications. This brighter signalimproves the accuracy of such sequencing-by-synthesis applications byreducing the number of incorporation events that go undetected. Inaddition, since each bead is joined to multiple phospholink nucleotides,each bead can be recycled through multiple incorporation events, thusreducing the amount of reagents needed in a particularsequencing-by-synthesis reaction.

In one aspect, the labeled phospholink nucleotides of the invention areutilized in polymerase reactions isolated within extremely smallobservation volumes, such as zero-mode waveguides, as described forexample in U.S. Pat. Nos. 6,917,726 and 7,056,661, each of which ishereby incorporated by reference in its entirety for all purposes, andin particular for teaching related to zero-mode waveguides andsequencing applications utilizing such zero-mode waveguides.

Sequencing Applications Utilizing Flow Cells

In one aspect, the phospholinked nucleotides of the present inventioncan be used in conjunction with a sequencing system that utilizes a flowcell. In such an aspect of the invention, fluorescent bead-labeledphospholinked nucleotides and a microfluidic flow cell are used todetect incorporation of labeled substrates into the nascent strand. Inan exemplary embodiment, a polymerase is immobilized at a stationarylocation in the flow cell relative to the remaining solution, which hasa constant fluid flow velocity. In a further embodiment, the polymeraseis immobilized on a surface of the flow cell and a solution containingbead-labeled phospholink nucleotides are flowed over the polymerase at aconstant velocity. The polymerase will temporarily retain a bead-labeledphospholink nucleotide complementary to a nucleotide of the templatestrand. This temporary retention of the labeled phospholink nucleotidewill be detectable relative to the remaining solution moving at aconstant fluid flow velocity around the polymerase. Thus, the residencetime in such a system is not measured by the absence or presence of alabel in a detection volume, but by the intermittent discontinuation ofmovement of the label as the phospholink nucleotide is held in theactive site of the polymerase.

In one embodiment, four differentially labeled phospholink nucleotidesare used in such flow cell reactions, and a complementary base of thetemplate will be identified by identifying a temporarily retained bead.

In a further embodiment, different nucleotides will be labeled withbeads comprising the same fluorophore, but the size of the bead willdiffer with regards to the identity of the nucleotide. For example,adenosine polyphosphates can be linked to smaller beads, whereasguanosine polyphosphates are linked to larger beads. The diffusionproperties before and after retention to the polymerase will thusdiffer, and the residence time combined with the diffusion kinetics ofthe temporarily retained bead-labeled phospholink nucleotide willprovide the identity of the phospholink nucleotide (and thus thecomplementary nucleotide on the template nucleic acid). Different sizesof beads will also differ by the number of encapsulated fluorophores,corresponding to different relative brightness, which can further aid indistinguishing the different nucleotides coupled to beads of differentsizes.

In a still further embodiment, detection of the temporarily retainedbead is accomplished using imaging methods known in the art, for exampletotal-internal reflection fluorescence (TIRF) microscopy.

Nucleic Acid Sequencing Systems

In one aspect, the labeled phospholink nucleotides of the invention areused in a nucleic acid sequencing system. Such a system can includeelements for conducting a sequencing-by-synthesis reaction in a singlemodule or device, or in a series of connected modules or devices. In afurther aspect, such a nucleic acid sequencing system can include a bodystructure comprising a plurality of microfluidic channels. Such a systemcan further include a source of one or more template nucleic acid, whichtemplate nucleic acid source is capable of being fluidly coupled to atleast a first one of the microfluidic channels and a source of one ormore sequencing reagents, which sequencing reagent source is capable ofbeing fluidly coupled to the at least first microfluidic channel. In astill further aspect, the source of the one or more sequencing reagentcan comprise a set of at least four differently labeled phospholinknucleotides of the invention, which, as discussed further herein, can inone embodiment include a fluorescent bead. In one exemplary embodiment,the system further includes a fluid flow controller that flows thetemplate nucleic acid and the one or more sequencing reagents intocontact in the at least first microfluidic channel, whereby one or moresequencing product is formed. In a still further embodiment, a detectordetects the one or more sequencing product, thereby determining at leasta subsequence of the template nucleic acid.

Kits

The present invention further provides kits useful for exploiting thelabeled phospholink nucleotides described herein in a number ofapplications. In a first aspect, such kits will generally include thelabeled phospholink nucleotides of the invention packaged in a fashionto enable their use, and preferably, a set of at least four differentphospholink nucleotides of the invention, namely those that areanalogous to A, T, G and C, where each bears a detectably differentlabel to permit its individual identification in the presence of theothers. Depending upon the desired application, the kits of theinvention may further include additional reagents, such as enzymes(including polymerase enzymes) for performing template dependentsynthesis reactions employing phospholink nucleotides of the invention,a control sequence, and other reagents, including buffer solutionsand/or salt solutions, including, e.g., divalent metal ions (such asMg²⁺, Mn²⁺ Ca²⁺, Co²⁺, Ba²⁺, Sr²⁺ and/or Fe²⁺), and standard solutions(such as dye standards for detector calibration). Such kits can alsoinclude instructions for use of the phospholink nucleotides and otherreagents in accordance with the desired application methods, e.g.,nucleic acid sequencing, and the like.

EXAMPLES Example 1 Coupling of Nanosphere Label to PhospholinkNucleotide

As illustrated in FIG. 1, a nanosphere with a surface derivatized withcarboxyl groups was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form an intermediate aceticN′-(3-(dimethylamino)propyl)-N-ethylcarbamidic anhydride compound,which, when contacted with a nucleoside polyphosphate, will result in abond between the terminal phosphate of the thymine hexaphosphate and theactivated functional group of the nanosphere. In the case of theillustration in FIG. 1, the nucleoside polyphosphate is a thyminehexaphosphate, but it will be appreciated that any nucleosidepolyphosphate could be used in accordance with this method.

In one exemplary embodiment, 1.68 μM 25 nm micromod Red nanoparticles(micromod Partikeltechnologie GmbH, Rostock, Germany) and 168 μMNH₂-PEG₁₂-15x-dC6P (100 fold excess) were mixed in 25 mM MES buffer.0.37 mg of EDC (5000 fold molar excess) were added while vortexing thesolution, followed by incubation for one hour at room temperature.

Optionally, NHS or sulfo-NHS can be included in the reaction (e.g., alsoat 5000 fold excess) to stabilize the amine-reactive intermediate byconverting it to an amine-reactive Sulfo-NHS ester, thus increasing theefficiency of EDC-mediated coupling reactions (see Grabarek, Z. andGergely, J. (1990), Analyt Biochem, 185:131-135 and Staros and Swing(1986) Analyt Biochem, 156: 220-222, each of which is herebyincorporated by reference in its entirety for all purposes and inparticular for all teachings related to coupling reactions as describedabove.

Example 2 Determining Spacing Necessary Between Bead Label andNucleotide

In order to determine the optimal distance between the bead and thenucleoside polyphosphate, multiple different linkers were tested in anincorporation reaction. FIG. 3 displays the results from one suchreaction. Of the three different linkers tested (A, B and C), only thenucleotide attached to a bead through linker C was successfullyincorporated by the polymerase. The structures of the three differentlinkers A, B, and C are illustrated in FIG. 2A, B, and C respectively.In these particular experiments, the polymerase used was Φ29 DNApolymerase.

The following reagents were mixed (to final concentrations): 50 mM ACESbuffer, pH 7.1, 75 mM potassium acetate, 5 mM DTT, 0.7 mM manganeseacetate, 100 nM circular single-stranded, primed DNA template, and Φ29DNA polymerase. The mixture was incubated for 10 minutes on ice, andthen nucleotides and bead-conjugated nucleotides were added asappropriate to a 5 μM final concentration. The mixture was thenincubated for 15 minutes at room temperature and then run on an 0.8%agarose gel with a 1 kb molecular weight marker as a calibration lane.The gel was stained with SybrGold nucleic acid stain and then imaged.Lanes AF, BF and CF in FIG. 3 depict absence of DNA polymerization whenthe 7th flowthrough (see FIG. 5) is used instead of the bead-conjugatednucleotide, proving the absence of free nucleotides in the bead samplesolution.

Example 3 Diffusion Time of Particles in Zero-Mode Waveguides

FIG. 4 shows the results of experiments measuring the diffusion times ofunbound beads and beads linked to phospholink nucleotides. FIG. 4A showsthe difference in diffusion times between the plain beads and the beadslinked to adenosine tetraphosphate (A488-dA4P) in a zero-mode waveguide(ZMW) of with a diameter of 80 nm. FIG. 4B shows the results of similarmeasurements conducted in a ZMW with a diameter of 120 nm. Theseexperiments show that the diffusion time decay for both the bare beadsand the bead-labeled phospholink nucleotides are both complete between 1and 10 milliseconds, which allows for a temporal differentiation betweendiffusion and incorporation events. Polymerase incorporation generallyoccurs in the 10 to 100 ms time scale.

Example 4 Purification Bead-Labeled Phospholink Nucleotides

In order to avoid incorporation of nucleotides that are not labeledaccording to the present invention, in one embodiment the inventionprovides methods for purifying bead-labeled phospholink nucleotides.

One way to purify the labeled nucleotides is through repeated washingand centrifugation through a 100 kDa MW cutoff membrane. The flowthroughabsorbance can be measured to monitor the purity.

The results of one such experiment are provided in FIG. 5. FIG. 5A showsthe flowthrough absorbance as a function of number of washes. FIG. 5Bshows the absorbance spectrum of three different types of bead-labelednucleotides. The peak on the right of FIG. 5B represents the absorptionspectrum of the bead-encapsulated fluorescent dye, and the peak andshoulder on the left side of the graph correspond to both absorption offluorescent dye and nucleotide. The arrow in FIG. 5B points to thedifference in absorbance between unconjugated and nucleotide-conjugatedbeads. Excess absorbance in this area indicates the presence ofbead-conjugated nucleotides in the sample.

The difference between the absorbance of “bare” particles to thosejoined to phospholink nucleotides can provide a quantitative estimate ofthe nucleotide/bead ratio, using the maximum absorption wavelengths forboth the dye and the nucleotide. Table I provides exemplary data forcalculating such a ratio. For example, the absorbance of the stockparticles can be measured at two different wavelengths (“A565 of stock”and “A267 of stock”). The absorbance of particles joined to phospholinknucleotides can be measured at the same two wavelengths. In Table I, thephospholink nucleotides measured are deoxythymine hexaphosphate analogsjoined to beads through a “uMod-PEG11-15x” linker, the structure ofwhich is shown in FIG. 2C. using the dye absorbance to calculate theconcentration of beads in the two samples, and the correspondingexpected absorbance at 267 nm (the maximum absorbance of thenucleotide), the excess absorbance at 267 nm for thenucleotide-conjugated sample is used to calculate the concentration ofnucleotides in the sample (using the known extinction coefficient fordTTP). The ratio between the concentration of nucleotide toconcentration of beads yields the number of nucleotides per bead.

TABLE I Particle stock concentration (from manufacturer) 5.50E+15particles/ml particle stock dilution 0.5 particle stock concentration4.58 uM A565 of stock 0.366 A267 of stock 0.135 A565 ofuMod-PEG11-15x-dT6P 0.701 A267 of uMod-PEG11-15x-dT6P 0.324uMod-PEG11-15x-dT6P particle concentration 8.78 uM corresponding A267from that concentration 0.259 A267 from dT6P on uMod-PEG11-15x-dT6P0.065 concentration of dT6P on uMod-PEG11-15x- 68.16 uM dT6P ratio ofdT6P/particles 7.76

Example 5 Flat-Glass Photodamage Assay

The effects of photodamage on bare particles (also referred to herein as“beads” or “nanospheres”) and on particles joined to phospholinknucleotides was measured in a flat-glass photodamage assay, in which DNApolymerase/DNA template complexes are immobilized on a glass surface,and irradiated with laser illumination while DNA synthesis is takingplace with fluorescence-labeled or bead labeled nucleotides. Thereafter,the presence or absence of active DNA polymerase is probed by DNAsynthesis using a base-linked nucleotide (e.g. ChromaTide-Alexa-Fluor488-dUTP, Invitrogen Corp., Carlsbad, Calif.), and subsequentbright-field microscopy imaging of the surface. Undamaged polymeraseincorporates the base-linked nucleotide into DNA, yielding fluorescencesignal from the surface, whereas polymerase damaged by laserillumination during the synthesis period with bead-conjugated analogwould be inactive, thereby rendering the illuminated region dark afterthe chromatide “development” step. No damage was seen for either thebare particles or for particles joined to phospholink nucleotides underreaction conditions in which oxygen was removed (e.g., by the use ofoxygen scavenging systems known in the art, such as the glucoseoxidase/catalase system, or the protocatechuic acid deoxygenase system).Damage was observed in oxygen conditions at 5 μW/μm², but thebead-labeled nucleotides were 10 times brighter than A532 and 20 timesbrighter than A568. This drastically improved brightness means that lesslaser power can be used in sequencing reactions, resulting in lessdamage to the polymerase.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. Unless otherwise clear from the context or expresslystated, any concentration values provided herein are generally given interms of admixture values or percentages without regard to anyconversion that occurs upon or following addition of the particularcomponent of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entirety for all purposes.

1. A composition, comprising a labeled phospholink nucleotide, whereinsaid labeled phospholink nucleotide comprises a structure that hasformula:

wherein B is a nucleobase; S is a sugar moiety; L is a bead comprisingan optically detectable property; R₁ is selected from oxygen and sulfur;R₂ is a linker moiety; and n is an integer selected from 0 to
 9. 2. Thecomposition of claim 1, wherein said linker moiety is a member selectedfrom substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, andsubstituted or unsubstituted heterocycloalkyl.
 3. The composition ofclaim 1, wherein said linker moiety comprises poly(ethylene glycol). 4.The composition of claim 1, wherein said bead is a latex bead.
 5. Thecomposition of claim 1, wherein said bead is a fluorescent bead.
 6. Thecomposition of claim 1, wherein said bead comprises a fluorophore. 7.The composition of claim 6, wherein said bead is encapsulated in anorganic or inorganic material.
 8. The composition of claim 6, whereinsaid bead is encapsulated in a polymeric material.
 9. The composition ofclaim 8, wherein said polymeric material is a member selected frompolyethylene glycol and alginate.
 10. A composition comprising amicrofluidic flow cell, said composition further comprising: a. anucleic acid polymerase immobilized on a surface of a substrate, whereinsaid substrate is contained within said microfluidic flow cell; and b.at least four differentially labeled nucleotides, wherein said labelednucleotides comprise beads comprising a fluorophore.
 11. A method ofdetermining an identity of a nucleotide in a template nucleic acidsequence, said method comprising: a. providing said template nucleicacid sequence complexed with a polymerase enzyme capable of templatedependent synthesis of a complementary nascent sequence as a firstcomplex; b. contacting said first complex with a labeled phospholinknucleotide, wherein said labeled phospholink nucleotide comprises a beadcomprising at least one fluorophore, and wherein said labeledphospholink nucleotide is complementary to a known nucleotide; and c.detecting whether said labeled phospholink nucleotide is incorporatedinto said nascent sequence, wherein incorporation of said labeledphospholink nucleotide is indicative that said complementary nucleotideis in a position in the template nucleic acid that is being processed bythe polymerase enzyme.
 12. The method of claim 11, wherein said firstcomplex is contained in a microfluidic flow cell, and wherein saiddetecting step comprises detecting a temporary retention of said bead ata stationary location of said flow cell.
 13. The method of claim 12,wherein said microfluidic flow cell comprises a surface, and whereinsaid polymerase of said first complex is immobilized on said surface ofsaid microfluidic flow cell.
 14. The method of claim 11, wherein saidfirst complex is contained in a zero-mode waveguide.
 15. The method ofclaim 11, wherein said bead comprises multiple fluorophores.
 16. Themethod of claim 15, wherein said detecting step comprises detecting asignal from said bead comprising multiple fluorophores, and wherein saidsignal from said bead has an improved signal relative to a beadcomprising a single fluorophore.
 17. The method of claim 11, whereinmultiple nucleotides are attached to said bead.
 18. The method of claim17, wherein subsequent to said detecting step, said method furthercomprises cleaving said bead from said phospholink nucleotide.
 19. Themethod of claim 18, wherein said providing, contacting, detecting andcleaving steps are repeated a number of times to identify a desirednumber of nucleotides in said template nucleic acid sequence.
 20. A kitcomprising at least four differently labeled nucleotide analogs eachcomprising said structure of said composition of claim 1, a polymeraseenzyme source, and instructions for using said composition with saidpolymerase enzyme in template directed nucleic acid synthesis.
 21. Anucleic acid sequencing system, comprising: a body structure comprisinga plurality of microfluidic channels; a source of one or more templatenucleic acid, which template nucleic acid source is capable of beingfluidly coupled to at least a first one of the microfluidic channels; asource of one or more sequencing reagent, which sequencing reagentsource is capable of being fluidly coupled to the at least firstmicrofluidic channel; wherein the source of one or more sequencingreagent comprises a set of at least four differently labeled nucleotideanalogs, wherein said labeled nucleotide analogs comprise a fluorescentbead, and a fluid flow controller that flows the template nucleic acidand the one or more sequencing reagents into contact in the at leastfirst microfluidic channel, whereby one or more sequencing product isformed, wherein a detector detects the one or more sequencing product,thereby determining at least a subsequence of the template nucleic acid.